Von Recklinghausen neurofibromatosis (NF1), one of the most common human autosomal dominant conditions, is characterized by cafe-au- lait spots, neurofibromas, Lisch nodules of the iris, and a variety of other manifestations. This locus has one of the highest spontaneous mutation rates recorded, about 10-4/allele/generation. In the three short years since the submission of our original proposal to initiate linkage studies in NF1, the gene has been mapped using RFLP markers to the proximal long arm of chromosome 17, through the efforts of our group and others. We now propose additional molecular approaches which should lead to the cloning and characterization of the NF1 gene. Specifically, we intend to refine the linkage analysis of our 21 NF1 families using multipoint methods, and to construct ordered genetic maps of less than 1 cM resolution by subjecting individual sperm to amplification of marker loci using the polymerase chain reaction (PCR). We will also construct a complete physical map of proximal 17q using pulsed field gel electrophoresis and libraries containing DNA fragments which include a rear restriction site (so-called linking libraries). Besides allowing the connection of parts of the physical map, these also provide entry points into chromosome jumping libraries. Importantly, a powerful set of somatic cell hybrids is available to allow the construction and mapping of such linking clones to the desired region of chromosome 17. A major question with this "reverse genetics" approach is how to identify the gene itself. Fortunately, we have available cell lines from two NF1 patients who carry balanced translocations involving 17q11. It is highly likely that the NF1 gene is disrupted by these translocations, so that identification and cloning of the breakpoints should allow cloning of the NF1 gene itself. This should be eminently achievable using the physical mapping approach combined with chromosome jumping. Once the breakpoints are cloned, the NF1 transcript will be identified by screening of appropriate cDNA libraries, and its tissue distribution studied. The genetic basis of NF1 will be investigated by analysis of this locus and its mRNA product in affected individuals. Finally, we will attempt to define the function of the normal gene by DNA transfer experiments into cultured cell lines and transgenic mice.